Ultrafast Excited-State Dynamics of Cytosine Aza-Derivative and

Publication Date (Web): March 23, 2017. Copyright © 2017 American ... (J.C.)., *E-mail: [email protected] (B.J.)., *E-mail: [email protected] (Y...
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Ultrafast Excited-State Dynamics of Cytosine Aza-Derivative and Analogues Zhongneng Zhou, Xueyao Zhou, Xueli Wang, Bin Jiang, Yongle Li, Jinquan Chen, and Jianhua Xu J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.6b12290 • Publication Date (Web): 23 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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Ultrafast Excited-State Dynamics of Cytosine AzaDerivative and Analogues Zhongneng Zhou1, Xueyao Zhou2, Xueli Wang1, Bin Jiang2,*, Yongle Li3,*, Jinquan Chen1,*, Jianhua Xu1

1

State Key Laboratory of Precision Spectroscopy, East China Normal University,

3663 North Zhongshan Road, Shanghai 200062, China; 2

Department of Chemical Physics, University of Science and Technology of China,

Hefei 230026, China; 3

Department of Physics, International Center of Quantum and Molecular Structures,

Shanghai Key Laboratory of High Temperature Superconductors, Shanghai University, Shanghai 200444, China;

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Abstract Excited state dynamics of 5-azacytosine (5-AC), 2,4-diamino-1,3,5-triazine (2,4-DT), and 2-amino-1,3,5-triazine (2-AT) were comprehensively investigated with steady state absorption, fluorescence and femtosecond transient absorption measurements. Time-dependent density functional theory (TDDFT) calculations were performed to help assign the absorption bands and understand the excited state decay mechanisms. The experimental results of excited singlet state dynamics for 5-AC, 2,4-DT and 2-AT with femtosecond time resolution were reported for the first time. Two distinct decay pathways, with ~1 picosecond and tens of picosecond lifetimes, were observed in 5-AC. Only one decay pathway with 17 picosecond lifetime was observed in 2,4-DT while an emissive state was found in 2-AT. TDDFT calculations suggest that 5-AC has a dark nπ* (S1) state below the first allowed ππ* (S2) state, which leads to the ultrafast decay of the ππ* state. In 2,4-DT, there is no dark nπ* state below the bright ππ* (S1) state and the 17 picosecond lifetime is assigned to the relaxation from the ππ* (S1) state to ground state. Two dark nπ* states (S1 and S2) were found in 2-AT which exhibits much more complex excited state dynamics compared with the other two. Photoluminescence in 2-AT has been confirmed to be fluorescence emission from its bright ππ* (S3) state. Our results strongly suggest that electronic structures are very sensitive to the substitution on the triazine ring and that the photophysical properties of nucleic acid analogues depend highly on their molecular structures.

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1. Introduction The appearance of ribonucleic acids (RNA) was the start of life due to its information storage capability and enzymatic functional properties.

1-5

Thus, revealing the origin

of RNA is essential for understanding the origin of life in the RNA world theory. In the photophysical and photochemical aspect, it is generally convinced that the canonical nucleobases are favored to serve as chromophores in RNA due to their excellent photo-stability which is reflected by their ultrafast excited state dynamics.6-11 However, many studies have suggested that the canonical nucleobases which common to today’s biology may have been preceded by alternative bases during the chemical evolution that took place before the RNA world.12-17 Biological modification18 and total chemical synthesis

19-21

have provided large variety of minor

nucleosides which could be possible ancestor nucleobases for prebiotic RNA. By studying the excited state dynamics of those nucleic base derivatives we hope to learn the impact of photo-stability which may have led to the selection of today’s canonical bases. Families of heterocyclic compounds, which are driven from the same type of starting materials as the canonical nucleobases, can be envisaged to be the primordial nucleobase alternatives. Triazine derivatives functionalized through their ring nitrogen substitution or addition can mimic both pyrimidine and purine, which possess the strong

prebiotic

relevance.22

Eschenmoser et

al.

have

demonstrated

that

2,4-diaminotriazine in dipeptidic oligomers shows strong base-pairing in aqueous solution, which is the constitutional and physicochemical prerequisites of potentially 3 ACS Paragon Plus Environment

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primordial oligomers.17 Meanwhile, Menor-Salvan et al. have demonstrated the successful syntheses of triazine in ice, which further supports the possible presence of these compounds in the prebiotic environment.23 Among all the triazine compounds, 1,3,5-triazines have shown significant difference in the excited state dynamics compared with canonical nucleobases. Kobayashi et al. first reported the drastically different excited state relaxation mechanism of 6-azauracil24 and then extended their study on 8-azaadenine, 5-azacytosine and 8-azaguanine.25 Gobbo and their collaborators have calculated the relaxation mechanisms of 8-azaadenine and 6-azauracil with CASPT2//CASSCF, and have revealed their unique conical intersections for nonradiative decay.26-27 Wierzchowski reviewed excited state photon transfer phenomenon including phototautomerism among the fluorescent nucleic acid base analogues, such as 8-aza-7-deazaadenine, 8-azaxanthine, 8-azaisoguanine et al. in 2010.

28

Later, Pollum

and co-workers reviewed steady state absorption property and excited state dynamics of the aza-bases for their important and potential biological applications.29 These aza-base analogues usually have a slightly red-shifted absorption spectra compared with canonical bases and exhibit weak fluorescence and no phosphorescence at the room temperature. The excited state dynamics property can be divided by two groups of aza-bases. One group consists of 6-azauracil and 8-azaadenine which have high triplet yield. The other group compounds such as 8-azaguanine and 5-azacytosine have very low triplet formation yield. They concluded that the 1nπ* state play an important role in modifying the photochemistry of aza-bases. However, most 4 ACS Paragon Plus Environment

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literatures were focused on the triplet state properties of aza-bases and nanosecond time resolution techniques were used. Recently, Hua et al. studied the methyl- and aza-substituent effects on nonradiative decay mechanisms of uracil using femtosecond transient absorption spectroscopy. They have

revealed that C6 and C5

methyl-substitution on uracil inhibits the 1ππ* - 1nπ* channel and improves the direct internal conversion from 1ππ* to S0, while aza-substitution at the C6 position blocks the direct internal conversion channel and leads to the major decay of the 1ππ* state to 1

nπ* state.30-31 Zhang et al. studied the excited state dynamics of melamine and its

lysine derivatives using femtosecond UV and mid-IR transient absorption techniques, proposing the observed 13 ps excited state lifetime could come from the nπ* state.32 However, full photophysical properties of the 1,3,5-triazine compounds have not been investigated thoroughly and such study will provide further insights into the origin of life in the RNA world. In this work, we studied the excited state dynamics of 5-azacytosine (5-AC), 2,4-diamino-1,3,5-triazine

(2,4-DT)

and

2-amino-1,3,5-triazine

(2-AT)

via

femtosecond transient absorption. The main reason for choosing these three derivatives is their structure similarity to cytosine as displayed in Figure 1. In particular, 5-AC was developed as antitumor agents and has been applied to treatments of both childhood and adult leukemias.33-37 Kobayashi and co-workers studied 5-AC by nanosecond laser flash photolysis and observed no triplet state formation.25 They claimed that the absence of a dark nπ* state below the first allowed ππ* state leads the low yield of intersystem crossing (ISC) process in 5-AC. On the 5 ACS Paragon Plus Environment

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other hand, Giussani et al. calculated the excited state potential energy surface of 5-AC and found a conical intersection (CI) between a nπ* state and the first allowed ππ* state.38 They have suggested that decay through this CI should account for the fast depopulation of the excited state in 5-AC. In a recent non-adiabatic ab initio molecular dynamics study, Borin et al. confirmed the influence of a low-lying nπ* state in relaxation mechanism of 5-AC and suggested that the initially populated ππ* state can decay to the nπ* state in tens of femtosecond timescale.39 They also showed that the intersystem crossing from the nπ* state to triplet state is possible with only ~10 ± 8% yield. In order to reconcile the contradiction among those previous studies, direct observation of the excited state dynamics of 5-AC with femtosecond time resolution is necessary. Meanwhile, comparative study on 2,4-DT and 2-AT would provide new insights into how amination/deamination of the triazine ring affects its nonradiative decay. The results in this work will help to understand the structural influence on the photochemistry properties of nucleobases and to promote the study of identification of promising RNA ancestors.

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Figure 1. Structure of cytosine, 5-azacytosine (5-AC), 2,4-diamino-1,3,5-triazine (2,4-DT) and 2-amino-1,3,5-triazine (2-AT).

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2. Experimental Methods 5-Azacytosine (5-AC) (98%) was purchased from Aladdin (Shanghai, China). 2,4-diamino-1,3,5-triazine (2,4-DT) and 2-amino-1,3,5-triazine (2-AT) (98%) were purchased from J&K Chemical Ltd. (Shanghai, China). All the samples were used as received without further purification. Samples were dissolved in the PBS buffer solution created with monosodium phosphate and disodium phosphate salts at a total salt concentration of 50 mM. The pH of the buffer was adjusted to be 7.4. All the water used in the experiment is 18.2 MΩ deionized water (Direct-Q3 UV, Merck Millipore). UV-Vis absorption spectra and emission spectra were measured using a double beam UV-Vis spectrometer (TU1901, Beijing Purkinje General Instrument Co. Ltd.) and steady state fluorescence spectrometer (FluoroMax-4, Horiba, Jobin Yvon) respectively. All experiments were carried out at room temperature. Transient absorption (TA) spectra were measured by a transient absorption spectrometer (Helios-EOS fire, Ultrafast System). Transient absorption kinetic traces were collected by a home-built pump-probe setup using a Ti:Sapphire laser system (Spitfire Pro, 800 nm, 35 fs, 1.7 mJ/pulse, and 1kHz repetition rate, Spectra-Physics). The 266 nm pump beam was generated from the third harmonic of the fundamental output (800 nm, ~300 mW) and the white light continuum (WLC) probe beam was generated by focusing a small portion (~0.5 mW) of 800 nm beam into a sapphire plate. The polarization between the pump and probe beams was set to be magic angle (54.7°). The kinetic traces were obtained by a home-built setup using a 8 ACS Paragon Plus Environment

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photomultiplier tube (PMT) detector (CR317, Hamamatsu) for higher sensitivity. The pump beam was chopped into 500 Hz by a synchronized chopper (MC2000, Thorlabs). The probe wavelength in the kinetic measurements was selected by a set of bandpass filters (10 nm). Signals from the PMT were sent into a lock-in amplifier (Model SR830, Stanford Research Systems) and recorded by data collection software written by LabWindows. The instrument response function (IRF) of this system was determined to be ~250 fs by measuring solvent responses under the same experimental conditions. Transient signals were corrected by removing the signal from two-photon ionization of water following a previously published procedure. 40-41 (see details in Supporting information (SI))

The kinetic signals were fit to sums of exponentials, ∑  exp  , where Ai is

the amplitude of the ith signal component, which decays exponentially with time constant τi. All fits were carried out using global fitting procedures in the IGOR Pro program version 6.37. (Wavemetrics Inc., Portland, OR) The coherent spike observed near t = 0 is excluded during data fitting process. To help the interpretation of experimental results, time-dependent density functional theory (TDDFT)42 calculations implemented in GAUSSIAN09 were performed. The ground state equilibrium geometries were optimized using the B3LYP functional43 with 6-311++G(d,p) basis set44, followed by the vertical excitation energies computed by TDDFT calculations. Solvent effects were approximated by means of Truhlar’s universal solvation model (SMD)45 based on the quantum mechanical charge density and a continuum model that describes solvent as a 9 ACS Paragon Plus Environment

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dielectric medium with surface tension. For excited states, in addition, minimum energy paths were recorded during the optimization as the steepest descent paths from the Franck-Condon geometries to the minima. For each system, 20 excited states were calculated for convergence and the absorption spectrum was simulated in terms of oscillator strengths and Gaussian functions centered at every transition using Multiwfn.46 More details and calculated results can be found in SI.

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3. Results and Discussion 3.1 Experimental and simulated UV-Vis absorption spectra

Figure 2. (a) UV-Vis absorption spectra of cytosine derivatives in buffer solutions (pH=7.4); (b) Calculated spectra by TDDFT with relative intensities of cytosine derivatives scaled to match the experimental counterparts. Scale factors for 2-AT and 2,4-DT are 2.01 and 0.88 respectively.

Figure 2 shows the experimental UV-Vis absorption spectra of the three cytosine derivatives in PBS buffer solution at pH=7.4, along with simulated spectra based on 11 ACS Paragon Plus Environment

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the vertical excitation energies and oscillator strengths from TDDFT calculations (see Table S1 and Figure S2 in SI). Overall, simulated spectra reproduce the measurements quite well with respect to the relative intensities and shapes. Consistent with our experimental observations, the calculated strongest absorption peaks for 5-AC, 2-AT, and 2,4-DT respectively, are in descending order of wavelength, though with a common blue shift of ~10 nm, which is actually below the typical error of TDDFT.47 These results validate that TDDFT captures the main characters of the electronic transitions for the systems of interest here in spite of the uniform overestimation of vertical excitation energies. Specifically, 5-AC shows two absorption bands with maxima at 201 nm and 240 nm, respectively. The weaker 240 nm absorption band has a shoulder on the red side which extends to ~260 nm. These features are well reproduced in the calculated spectrum, where the peak of both bands is at ~190 nm and ~230 nm respectively. The former is associated with two high-lying ππ* excited states with very strong oscillator strengths closed to ~0.5, whereas the latter arises from ππ* (S2) and ππ* (S3) states which are mixed with both HOMO-2 → LUMO and HOMO → LUMO transitions carrying obvious charge transfer character from the carbonyl group to the aromatic ring. Compared with the canonical nucleobase cytosine, 5-AC has no absorption peak at around 270 nm9, which has been assigned to the HOMO to LUMO transition in cytosine.48 Interestingly, the adsorption bands measured here in water shifts to the blue compared with that measured in acetonitrile by Kobayashi and coworkers.25 When the carbonyl group is replaced by an amino group at the C2 position on the 12 ACS Paragon Plus Environment

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triazine ring, 2,4-DT shows a clear absorption peak at 258 nm as well as a much stronger absorption peak at 205 nm. TDDFT estimates the absorption bands at ~240 and 196 nm, which can be attributed to two ππ* states S1 and S10 with the former being assigned to the HOMO → LUMO transition and the latter the HOMO-1 → LUMO+1 transition. Removal of the amino group at the C4 position, 2-AT shows a broader absorption peaked at 262 nm compared to 2,4-DT. Additionally, a strong absorption band with a maximum at 221 nm can also be seen in 2-AT. This band is very similar to the strong absorption band shown in 2,4-DT, but is redshifted by ~20 nm. Based on the TDDFT results, these two peaks are related to two ππ* states arising from HOMO→LUMO transition localized on the six-membered ring at 253 nm (S3) and HOMO → LUMO+1 transition at 208 nm (S6), respectively. It should be noted that excitation of both 5-AC and 2,4-DT in the spectral range from 250 to 270 nm resulted in no detectable emission in aqueous solution, indicating that effective nonradiative pathways should exist in these two compounds. For 2-AT, fluorescence emission can be detected under UV excitation (Figure S5), the significance of this observation will be discussed in more detail below.

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3.2 Femtosecond transient absorption spectra and kinetics of 5-AC 1.5 1.0

(a) 5-AC

0 ps 0.2 ps 0.4 ps 0.6 ps 0.8 ps 1.2 ps

(b)

1.2 ps 1.8 ps 2.6 ps 6.2 ps 11.9 ps 23.4 ps 83.3 ps

0.5 0.0 1.5

3

OD (x10 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.0

(c) 2,4-DT

(d)

-0.05 ps 0.3 ps 0.6 ps 0.8 ps 1.0 ps 1.5 ps

1.5 ps 4.1 ps 9.2 ps 19.3 ps 31.1 ps 83.3 ps

0.5 0.0 1.0

0.5

(e) 2-AT

(f) 0 ps 0.3 ps 0.4 ps 0.8 ps 1.2 ps

1.2 ps 1.9 ps 5.4 ps 14.5 ps 39.0 ps 79.2 ps

0.0 450 500 550 600 650 700

450 500 550 600 650 700

Wavelength(nm) Figure 3. Broadband transient absorption spectra of (a-b)5-AC, (c-d) 2,4-DT and (e-f) 2-AT at neutral pH with 266 nm excitation.

Femtosecond transient absorption (TA) measurements were carried out on the three triazine compounds in order to reveal the excited-state dynamics and the TA spectra are displayed in Figure 3. The pump excitation was performed to the red end of the absorption spectra (266 nm). As shown in Figure 3 (a-b), there is only one broad TA band with maximum at ~550 nm in 5-AC. This band rises within time resolution of

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our TA setup and then decays to zero in the next ~100 ps. This observation coincides with a previous nanosecond TA study by Kobayashi et al.25, suggesting that 5-AC has very low triplet state formation yield. Excited-state decay dynamics of 5-AC are very similar in our whole probe range and they can be globally fitted by a two-exponential decay function as shown in Figure 4(a). The lifetimes were determined to be τ1=1.5 ± 0.2 ps and τ2=15 ± 1 ps, respectively. It is known that TA signals at visible range from all five canonical nucleobases decay on a sub-picosecond time scale and these signals are believed to monitor the process of ultrafast depopulation of the initial excited states.6-8, 49-50 In the case of cytosine, theoretical calculations have predicted that conical intersections (CIs) are responsible for the ultrafast nonradiative decay from excited state to ground state.48, 51-55

Based on the calculated energies and oscillator strengths in this work (Table S1),

the 266 nm excitation is expected to drive 5-AC to its ππ* (S2) state. This result agrees with previous TDDFT calculations for 5-AC in acetonitrile by Kobayashi et al.25, in which the ππ* state was predicted to dominate in broad absorption band near 260 nm. Furthermore, ab initio calculations with both CASPT2//CASSCF and MRCIS method also show that 5-AC should populated the ππ* (S2) state under 266 nm excitation.38-39 We have carefully examined dynamics in the whole visible probe wavelengths and found that they are very similar as shown in Figure 4(a), suggesting that the dynamics at visible probe wavelengths are from the same transient species. Ma et al. reported broad TA spectra (280 to 600 nm) of cytosine as well as its N1 and C5 derivatives.

56

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broadly in the visible range while the nπ* state shows absorption in the UV range with different band features. Brister et al. investigated 2,4,6-triaminopyrimidine and discovered that the TA band features are quite different between the ππ* and nπ* even though both states absorb in visible region.57 In addition, the pKa value of 5-AC ring has been determined to be 3.52,58 which suggests that 5-AC should be in the neutral form under our experimental condition. Therefore, we assigned the broad transient absorption signal to the excited-state absorption of the singlet ππ* (S2) state in neutral 5-AC.

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(a) OD(a.u.)

5-AC (600 nm)

5-AC (550 nm)

5-AC (500 nm) -5

0

5

9

2

3

4

5

6

7 8

10

Time Delay(ps)

(b) OD(a.u.)

2,4-DT (700 nm) 2,4-DT (650 nm) 2,4-DT (600 nm)

-5

0

5

9

2

3

4

5

6

7 8

10

Time Delay(ps)

(c)

2-AT (475 nm)

OD(a.u.)

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2-AT (450 nm) 2-AT (425 nm)

-5

0

5

9

2

3

4

5

6

7 8

10

Time Delay(ps) Figure 4. The kinetics of (a) 5-AC, (b) 2,4-DT and (c) 2-AT at representative wavelengths. The circles are experimental data and the solid lines are best-fits. The corresponding fitting parameters can be found in Table 1. Two lifetimes are required to adequately fit the kinetic traces of 5-AC shown in Figure 4(a), suggesting the ππ* state undergoes fast depopulation via two distinct channels. Giussani and co-workers have done extensive calculations at CASPT2//

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CASSCF level and suggested that there could be two relaxation processes for 5-AC from the excited-state to ground state.38 Indeed, it was found that the initially populated bright ππ* state can evolve to either an nπ* state or the ground state directly through different CIs. The CI between the ππ* and nπ* state lies at 4.04 eV above the ground state minimum, which is quite close to the Franck-Condon (FC) region and is involved in the minimum energy path (MEP) of the ππ* state decaying from the FC geometry to (ππ*)min. This is in qualitative agreement with our TDDFT MEP as shown in Figure 5(a) presenting a (ππ*/ nπ*) CI in the vicinity of the FC region. This deactivation pathway further involves the relaxation from this CI to the (nπ*)min, followed by a small energy barrier of 0.18 eV before reaching the CI between the nπ* state and ground state.38 On the other hand, the system may decay to (ππ*)min with the energy of 3.69 eV above the S0min and overcome a 0.37 eV barrier before accessing the CI between the ππ* and ground state, representing the second deactivation pathway. These two distinct pathways correspond well to the observed bi-exponential decay of the broad transient absorption band in the visible range. Since the (ππ*/nπ*) CI is found to be much closer to the FC region38, as illustrated in Figure 5(a), we assign the 1.5 ps lifetime to the decay channel from the initially excited ππ* state to the nπ* state via the (ππ*/ nπ*) CI, followed by a fast relaxation to nπ*min and to ground state by the second CI between (nπ*)min and ground sate. Meanwhile, the remaining population in the ππ* state needs to relax to the ππ*min firstly and then overcome an energy barrier in order to reach the CI between ππ* and ground state. As a result, we believe that the 15 ps lifetime should be assigned to 5-AC decaying back 18 ACS Paragon Plus Environment

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to ground state directly from the ππ* state. It should be noted that calculated energy states mentioned here and afterwards are relative to the equilibrium of ground state, and their absolute energies should be taken with caution (See SI for more details about the energetics). Very recently, Borin et al. investigated gas phase relaxation dynamics of excited 5-AC by ab initio molecular dynamics (AIMD) simulations at the MRCIS/def2-SVP level.39 The computed energies for relevant singlet and triplet states of 5-AC were coincident well with their previous study.38 However, the conical intersection between the first allowed ππ* and ground state predicted in previous calculation38 was not obtained at the MRCIS level, possibly due to the difference between the two methods used in their simulations. According to the AIMD-results, the initially populated ππ* (S2) state will decay to the nπ* (S1) state via an ultrafast internal conversion with a time constant estimated to be 32 fs.39 Subsequently, the nπ* (S1) state relaxes to populate the ground state (S0), accompanied by a small portion of intersystem crossing to the triplet state. Noted that their calculations were performed in vacuum, the magnitude of the reported lifetime is likely underestimated. In this opinion, we believe that the proposed relaxation mechanism is coincident with our observed dynamics and the lifetime of the decay from the ππ* (S2) state to the nπ* (S1) state is 1.5 ps. On the other hand, the 15 ps lifetime observed in 5-AC matches well with the 17 ps lifetime observed in 2,4-DT. (Table 1) Since 2,4-DT have no nπ* state below

ππ* state, the 17 ps lifetime can only be assigned to the decay from ππ* state to ground state as we will discuss in section 3.3. Thus, we propose that there should be a 19 ACS Paragon Plus Environment

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CI between the ππ* (S2) and ground state38 should exist in 5-AC which accounts for the 15 ps decay in our experimental data. This may require further theoretical investigations to confirm. Due to the limitation of probe range and time window, we are not able to detect the intersystem crossing process from the nπ* (S1) state to triplet states in 5-AC as proposed by Borin et al. so far and we plan to discuss this process in a later publication.

Figure 5. Energy profiles of the ground and lowest three singlet excited states of 5-AC (a), 2-AT (b), and 2,4-DT (c) along the minimum energy path (MEP) from ground state minimum to the minimum of the first ππ* state in each system.

Many theoretical studies have modeled the nonradiative decay pathways in nucleobases and their derivatives.6,

24, 26-27, 59-61

The general consensus is that the

dynamics of nucleobases excited-states depend very sensitively on the potential energy landscape determined by molecular structure. In pyrimidines, deformation of the C5=C6 bond is essential to form the CIs between the ππ* and nπ* states or directly with the ground state. It has been pointed out that torsion of the ring along the

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C5=C6 bond could lead to an ethylenic type S1/S0 CI48, 61-64 and that is believed to account for the ultrafast excited-state dynamics of cytosine in condensed phase50, 65 The structure of 5-AC is identical to that of cytosine except for the substitution of carbon with nitrogen at the 5 position. Our TDDFT calculations indicate that the N5=C6 bond is shortened in 5-AC (1.30 Å) compared with C5=C6 bond in cytosine (1.37 Å)66 (Figure S1), which could hamper the torsion of C6 as well as the access to the CI between the ππ* state and ground state as proposed in previous studies.24, 26, 29 In fact, the predicted barrier by Giussani et al. between the ππ*min and ethene-like ππ*/gs CI is 0.37 eV, much higher than that in cytosine (0.1~0.15 eV).51, 67-68 The consequence is that the excited-state lifetime of 5-AC presents a ~40-fold increase compared with that of cytosine. Our observation agrees with previous studies in which substitution at C5 position not only increases the nonradiative decay lifetimes but also increases the fluorescence lifetimes in cytosine derivatives.69-70 Another effect of the N atom substitution in 5-AC is that it leads to the appearance of a low energy nNπ* state as seen in our TDDFT calculations and previously reported in literature.26, 38

In cytosine, most theoretical work predicts that both the nOπ* and nNπ* state should

have higher energy than the lowest ππ* state61, leaving the ultrafast decay from the ππ* state to ground state through CI to be the only decay pathway for cytosine. Although the existence of the lower energy nπ* (S1) state in 5-AC opens a channel to depopulate the initially excited ππ* (S2) state, restricting the torsion of N5=C6 bond has been proposed to cause a much slower decay of the excited state.24, 26, 29 It is known that an ultrashort excited-state lifetime, which improves the photo-stability, is 21 ACS Paragon Plus Environment

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a general property of canonical nucleobases which enhances stability in a harsh UV environment.11 In this aspect, 5-AC could be a hot spot for photodamage even though it has been demonstrated to prohibit DNA methylation and could be used in cancer therapy. 33-34, 37

Scheme 1. Proposed relaxation mechanism for (a) 5-AC, (b) 2,4-DT and (c) 2-AT. The colored arrows represent the deactivation pathways fully supported by the current TA data. The gray arrows represent the adopted plausibly pathways proposed in literatures.38-39

3.3 Femtosecond transient absorption spectra and kinetics of 2,4-DT and 2-AT Next we move on to the TA results of the other two chosen cytosine azaanalogues in this study. 2,4-DT has an amino group at C2 position replacing the carbonyl group in 5-AC (Figure 1). The pKa value of 2,4-DT has also been reported to be 5.0 and 8.7,71 suggesting that this molecule is presented in the neutral form in this study. The TA spectra of 2,4-DT in buffer solution (pH=7.4) are shown in Figure 3(c-d). Compared with that of 5-AC, the TA band of 2,4-DT shows a large red-shift. 22 ACS Paragon Plus Environment

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The TA signals are much stronger at 700 nm and there is a residual absorption band with much lower amplitude below 450 nm. The signals also build up within the instrument resolution and then decay to zero within the next 80 ps. The slightly negative TA band between 450 and 500 nm might come from the hydrated electron signal correction. (see Figure S4 and detail in SI). Thus, the excited state dynamics of 2,4-DT were examined in the red region (600 to 750 nm) where the TA signal is strong. The kinetic traces of 2,4-DT at selected wavelengths are illustrated in Figure 4(b). Only one exponential decay components was required to globally fit the kinetics from 600 nm to 750 nm and the lifetime was found to be 17 ± 1 ps.

Table 1. Best-fit parameters for transient absorption kinetic traces of 5-AC, 2,4-DT and 2-AT in buffer solutions. a

5-AC

2,4-DT

2-AT a

λdet (nm)

A1 (%)

τ1 (ps)

A2 (%)

τ2 (ps)

600

29

550

18

500

16

84

700

100

-

-

650

100

-

-

600

100

-

-

475

78

22

-

450

68

32

-

425

60

40

-

71 1.5 ± 0.2

17 ± 1

2.6 ± 0.2

82

15 ± 1

Amplitude percentage was calculated by  =  ⁄∑ | |

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performed for 2,4-DT previously. However, the structure of 2,4-DT resembles that of nucleobase cytosine but with C5 replaced by N5 and the oxygen atom connecting to C2 replaced by an amino group, as seen in Figure 1. Consequently, we can use the extensive computational and experimental knowledge of cytosine and its derivatives to provide some reference to the observed excited-state dynamics in 2,4-DT. Interestingly, our TDDFT results displayed in Figure 5b suggest that the absence of the carbonyl group at C2 position comparing to 5-AC turns the initially populated ππ* (S1) state in 2,4-DT into the lowest excited state. Our results agree with previous studies on 2,4-diaminopyrimidine, which has almost the same structure as 2,4-DT except for a carbon atom at 5 position.72-73 Therefore, we attribute the broad TA absorption band to the excited-state absorption of ππ* (S1) state given its large oscillator strength as listed in Table S1 while the vertical excitation energies of the first three singlet states are quite close to each other. The vanished TA absorption signals of 2,4-DT suggest that the initially populated ππ* (S1) state should recover to ground state completely within 80 ps. Based on our TDDFT results, the decay channel from ππ* to nπ* state via the CI close to the FC region, which is predicted to exist in 5-AC, should no longer exist in 2,4-DT. This is confirmed in our TA data since we only observed one decay component with 17 ps lifetime in 2,4-DT. Nachtigallová and co-workers have pointed out that highly efficient deactivation channels with C5=C6 torsion exist in 2,4-diaminopyrimidine even though amino substitution could block the C2 deformation and 1.3 ps excited-state lifetime was predicted in their calculations.72 In 24 ACS Paragon Plus Environment

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our study, the structure similarity of 5-AC and 2,4-DT as well as the highly consistent lifetimes indicate that the 17 ps lifetime may correspond to the decay from the ππ* (S1) state to the ground state in a similar way as that of 5-AC. Once again, the N5=C6 bond in 2,4-DT hinders the C6 twist, presumably causing higher barrier between the ππ*min and ethene-like ππ*/gs CI,24,

26, 29

thus leading to an ~35 times longer

excited-state lifetime of 2,4-DT compared with cytosine. Our findings are in line with a previous report where excited-state lifetimes of 2,4-diaminopyrimidine range from 10 ps to 1 ns.73 In general, we propose that after 266 nm excitation 2,4-DT could decay back to ground state via an ethene-like ππ*/gs CI as illustrated in Scheme 1(b). The same measurements were also carried out on 2-AT at neutral pH in buffer solution and the corresponding TA spectra are illustrated in Figure 3(e-f) while the dynamics are shown in Figure 4(c) for comparison. Previous study reported the pKa value of 2-AT should be 2.9 and 14.9,74 suggesting that 2-AT is in neutral form in buffer solution (pH=7.4) and the interference from the tautomer should not exist in our study. Again, the TA signals build up within the instrument resolution just like 5-AC and 2,4-DT. Different from 5-AC and 2,4-DT, 2-AT shows strong TA signal in the near UV region (600 nm). The dynamics also show remarkable difference when compared to the previous two compounds. It has been confirmed that there is a long-lived component in 2-AT even after correction of the solvent electron signal from two-photon ionization of water. Since the lifetime of the long-lived species is beyond our experimental setup limit, we have to use one exponential and a constant offset to 25 ACS Paragon Plus Environment

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fit the dynamics observed in 2-AT. The lifetime was found to be 2.6 ± 0.2 ps. Once again, there is no high level ab-initio excited state calculation for 2-AT. In the present work, TDDFT predicts that the first three singlet states lie in a small energy window. Since ππ* (S3) state has the largest oscillator strength, it is expected to be primarily populated upon the 266 nm excitation and there are studies show that compounds with structural similarity as 2-AT could exhibit broad absorption band of the ππ* state in the visible range.29-30, 56-57, 75 Thus, we assign the broad TA signal to excited-state absorption of the ππ* (S3) state as the case in 5-AC and 2,4-DT. However, there are two nπ* states (S1 and S2) below the initially excited ππ* state, making the excited state dynamics much more complex than the other two compounds in this study. Due to the time window limitation of our current experimental setup, it is difficult to identify full decay pathways for the excited state dynamics of 2-AT. Thus, only plausible relaxation mechanism can be concluded for 2-AT. Fortunately, we can still compare it with molecules which have highly structural similarity to 2-AT. The first compound we need to address is 4-aminopyrimidine, which has almost the same structure as 2-AT except for a carbon atom at 5 position. Several theoretical studies have pointed out that 4-aminopyrimidine could reach a S1/S0 CI mainly via puckering at the C6 carbon atom and the excited state lifetime is estimated to be ~1 ps.72, 76-77 This scenario may not exist in 2-AT since we have observed long-lived TA signal. As discussed above, Borin and coworkers have proposed a complete relaxation mechanisms for 5-AC recently.39 Since the similarity of the structure between 5-AC 26 ACS Paragon Plus Environment

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and 2-AT, such relaxation mechanism could provide significant reference for interpreting the complex deactivation pathways of 2-AT. For 5-AC, the initially populated ππ* state will decay to the low-lying nπ* state through a CI. In the case of 2-AT, TDDFT calculated vertical excitation energies predict that there are two nπ* states (S1 and S2) below the initially excited ππ* (S3) state (4.91 eV) with the energies of 4.76 eV and 4.88 eV respectively (Table S1). Figure 5(b) displays the energies of the low-lying singlet states for 2-AT along the MEP from FC geometry to ππ*min. It is clear that the ππ* state cross with two nπ* states almost in the FC region, implying fast transition from ππ* to nπ* could happen via the associated CIs. Therefore, we assign the 2.6 ps lifetime observed in our TA experiment to the internal conversion from the initial ππ* state to the lower nπ* states. However, it is difficult for us to identify which nπ* (S2 or S1) state accounts for the major deactivation pathway in the current stage. According to the oscillator strength, it might be assumed that the nπ* (S1) state is dominant compared with nπ* (S2) state. Besides of such rough assumption, we will systematically investigate the detail of the relaxation mechanism of 2-AT in our further study. Photoluminescence was detected in 2-AT with 266 nm excitation covering the range from 300 nm to 500 nm. (Figure S5) We have carried out deoxygenation experiment with 2-AT. Only bare enhancement of the emission intensity was observed after the solution was saturated with nitrogen gas, indicating that photoluminescence of 2-AT is fluorescence emission. The excitation spectra of 2-AT (Figure S6) match the absorption spectra very well, suggesting that the fluorescence is from the bright 27 ACS Paragon Plus Environment

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ππ* (S3) state. This observation indicates that there is also a radiative decay pathway for the ππ* (S3) state in 2-AT. There is a long-lived component with lifetime longer than our instrument detection range (1000 ps) observed in 2-AT. Due to the limitation of time window and detection range in the current TA setup, it is difficult to give a conclusive assignment to the origin of the long-live species. Here, we will discuss plausible explanation for this species. Hare et al. have investigated the excited state dynamics for several pyrimidine bases in aqueous solution and proposed two distinct pathways for the initially populated ππ* state.78 They discovered that one pathway involves an nπ* state as an intermediate state and the lifetime of the nπ* state is tens to hundreds of picosecond. Ma et al. reported broadband TA spectra cytosine and derivatives and located a TA band responsible for nπ* state absorption in 300 to 400 nm range. The lifetime accounting for the dark nπ* state were reported to be several to hundreds of picoseconds depending on the solvents and substitutions.

56

Brister and co-workers

reported that the excited bright ππ* state in 1-cyclohexyluracil can internally convert to an nπ* state which has the TA absorption band centered at ~410 nm.75 Since the structure of 2-AT is similar to those molecules discussed above and the ππ* (S3) state in 2-AT can convert to one of the nπ* states in 2.6 ps, we suggests that the long-lived TA signal might contribute from the nπ* state. Another possible assignment of the long-live component in 2-AT is to the triplet states. Our TDDFT results show that there are four triplet states (T1-T4) with energy lower than the two nπ* states. (Table S1) According to the latest calculation for 5-AC by Borin and coworkers, the 28 ACS Paragon Plus Environment

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subsequently populated nπ* (S1) state can either decay back to the ground state or populate the triplet state through intersystem crossing.39 Triplet states have been discovered in several DNA aza analogues and the absorption peaks of those states were usually under 400 nm.29 Kobayashi and coworkers have reported the nanosecond excited dynamics of 6-azauracil, 6-azauridine and 8-azaadenine.24-25, 79 For 6-azauracil, the relatively intense triplet absorption band was located at 320 nm with the decay rate constant estimated to be (5.3 ± 0.2)×106 s-1. The triplet quantum yield and singlet oxygen yield of 6-azauracil were reported to be 0.93 and 0.63 respectively, which are quite similar with those of 6-azauridine. The triplet lifetime and singlet oxygen yield of 8-azaadenine were reported to be 240 ± 5 ns and 0.15 ± 0.02. Thus, it is reasonable that there could be long-lived TA peaks show up in the blue side of our probe range if triplet states were populated in 2-AT. Further investigations such as transient absorption with a longer time window and wider spectral range, fluorescence lifetime measurements and high-level calculations, both static and dynamical, are necessary to reveal the full excited state relaxation mechanism for 2-AT.

4. Conclusions Three cytosine aza- derivatives were studied by femtosecond transient absorption for the first time. Despite the structural similarity, the three triazine compounds exhibit quite different excited state dynamics in buffer solution. Replacing the C5 carbon atom with nitrogen atom leads to ~35-40 times longer excited state lifetimes in 5-AC and 2,4-DT compared with that of cytosine, possibly due to the hindered torsion of the 29 ACS Paragon Plus Environment

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C5=C6 bond that leads to access the CI between initial excited ππ* state and ground state.24, 26, 29 Meanwhile, a CI between the ππ* state and a low-lying nπ* state should account for the ultrafast excited state dynamics in 5-AC while no such CI was found in 2,4-DT. Further removing one amino group at C4 position made 2-AT the only fluorescence emissive molecule under UV excitation among these three compounds. Internal conversion from the excited ππ* state to one of the two low-lying nπ* states is observed in 2-AT. TDDFT calculation results in this study help to discuss the decay pathways qualitatively, but more advanced static and dynamical calculations are required in order to have a quantitative understanding of the excited state dynamics in these three triazine molecules. Overall, these results show that the electronic structures are strongly altered by nitrogen atom substitution in cytosine azaderivatives and the photophysics properties of nucleic acid base analogues are largely dependent on these structures. It is our hope that this work will stimulate more future investigations on the photophysics of the nitrogen substituted nucleic acids.

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ASSOCIATED CONTENT Supporting Information Optimized molecular structures and orbitals of three triazine derivatives, solvent electron correction process and steady state fluorescence of 2-AT. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *E-mail:

[email protected]

(J.C.);

[email protected]

(B.J.);

[email protected](Y. L.)

ACKNOWLEDGMENTS This study was funded by National Nature Science Foundation of China (No. 11674101 to J. C.; No. 21503130 and No. 11674212 to Y. L., No. 21573203 to B.J.). Y. L. is also supported by Young Eastern Scholar Program of the Shanghai Municipal Education Commission (QD2016021) and Shanghai Key Laboratory of High Temperature Superconductors (No. 14DZ2260700). The calculations have been done on the supercomputing system in the Supercomputing Center of University of Science and Technology of China. B. J. thanks Changjian Xie, Yingjin Ma, Guangshuangmu Lin for their helpful discussions.

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